Polynucleotides, vectors and host cells encoding LicB from streptococcus pneumonial

ABSTRACT

The invention provides licB polypeptides and polynucleotides encoding licB polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing licB polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/024,848, filed Feb. 17, 1998 now U.S. Pat. No. 5,962,295.

This application claims benefit of priority of U.S. ProvisionalApplication Ser. No. 60/033,807, filed Feb. 28, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to novel polynucleotides and polypeptides of the licB family,hereinafter referred to as “licB”.

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains which are resistantto some or all of the standard antibiotics. This phenomenon has createda demand for both new anti-microbial agents, vaccines, and diagnostictests for this organism.

Clearly, there exists a need for factors, such as the licB embodimentsof the invention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists to find ways to prevent,ameliorate or correct such infection, dysfunction and disease.

Certain of the polypeptides of the invention possess amino acid sequencehomology to a known licB protein. See NCBI non-redundant proteindatabase at gi|074967|pir∥C64128 and gi|1574380 (U32829); also see TIGRStreptococcus pneumonia sequences available from The Institute forGenome Research via the internet at http://www.tigr.org/, Entry 4179;and Weiser et al., Infect. Immun., 65(3), 943-50 (1997).

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel licB polypeptides by homology between the amino acidsequence set out in Table 1 [SEQ ID NO: 2 or 4] and a known amino acidsequence or sequences of other proteins such as licB protein.

It is a further object of the invention to provide polynucleotides thatencode licB polypeptides, particularly polynucleotides that encode thepolypeptide herein designated licB.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding licB polypeptides comprisinga sequence set out in Table 1 [SEQ ID NO:1 or 3] which includes a fulllength gene, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel licB protein from Streptococcus pneumoniae comprising the aminoacid sequence of Table 1 [SEQ ID NO:2 or 4], or a variant thereof.

As a further aspect of the invention there are provided isolated nucleicacid molecules encoding licB, particularly Streptococcus pneumoniaelicB, including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of licB and polypeptides encoded thereby.

In another aspect of the invention there are provided novel polypeptidesof Streptococcus pneumoniae referred to herein as licB as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of licB polypeptide encoded by naturally occurring alleles ofthe licB gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned licB polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing licBexpression, treating disease, assaying genetic variation, andadministering a licB polypeptide or polynucleotide to an organism toraise an immunological response against a bacteria, especially aStreptococcus pneumoniae bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to licB polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of the invention there are providedantibodies against licB polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided licB agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a licB polynucleotide or a licB polypeptide foradministration to a cell or to a multicellular organism.

In another embodiment of the invention there is provided a computerreadable medium having stored thereon a member selected from the groupconsisting of: a polynucleotide comprising the sequence of SEQ ID NO. 1or 3; a polypeptide comprising the sequence of SEQ ID NO. 2 or 4; a setof polynucleotide sequences wherein at least one of said sequencescomprises the sequence of SEQ ID NO. 1 or 3; a set of polypeptidesequences wherein at least one of said sequences comprises the sequenceof SEQ ID NO. 2 or 4; a data set representing a polynucleotide sequencecomprising the sequence of SEQ ID NO. 1 or 3; a data set representing apolynucleotide sequence encoding a polypeptide sequence comprising thesequence of SEQ ID NO. 2 or 4; a polynucleotide comprising the sequenceof SEQ ID NO. 1; a polypeptide comprising the sequence of SEQ ID NO. 2;a set of polynucleotide sequences wherein at least one of said sequencescomprises the sequence of SEQ ID NO. 1; a set of polypeptide sequenceswherein at least one of said sequences comprises the sequence of SEQ IDNO. 2; a data set representing a polynucleotide sequence comprising thesequence of SEQ ID NO. 1; a data set representing a polynucleotidesequence encoding a polypeptide sequence comprising the sequence of SEQID NO. 2.

A further embodiment of the invention provides a computer based methodfor performing homology identification, said method comprising the stepsof providing a polynucleotide sequence comprising the sequence of SEQ IDNO. 1 in a computer readable medium; and comparing said polynucleotidesequence to at least one polynucleotide or polypeptide sequence toidentify homology.

A further embodiment of the invention provides a computer based methodfor performing homology identification, said method comprising the stepsof: providing a polypeptide sequence comprising the sequence of SEQ IDNO. 2 in a computer readable medium; and comparing said polypeptidesequence to at least one polynucleotide or polypeptide sequence toidentify homology.

A further embodiment of the invention provides a computer based methodfor polynucleotide assembly, said method comprising the steps of:providing a first polynucleotide sequence comprising the sequence of SEQID NO. 1 in a computer readable medium; and screening for at least oneoverlapping region between said first polynucleotide sequence and asecond polynucleotide sequence.

A further embodiment of the invention provides a computer based methodfor performing homology identification, said method comprising the stepsof: providing a polynucleotide sequence comprising the sequence of SEQID NO. 1 in a computer readable medium; and comparing saidpolynucleotide sequence to at least one polynucleotide or polypeptidesequence to identify homology.

A further embodiment of the invention provides a computer based methodfor performing homology identification, said method comprising the stepsof: providing a polypeptide sequence comprising the sequence of SEQ IDNO. 2 in a computer readable medium; and comparing said polypeptidesequence to at least one polynucleotide or polypeptide sequence toidentify homology.

A further embodiment of the invention provides a computer based methodfor polynucleotide assembly, said method comprising the steps of:providing a first polynucleotide sequence comprising the sequence of SEQID NO. 1 in a computer readable medium; and screening for at least oneoverlapping region between said first polynucleotide sequence and asecond polynucleotide sequence.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to novel licB polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel licB of Streptococcuspneumoniae, which is related by amino acid sequence homology to licBpolypeptide. The invention relates especially to licB having thenucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1and SEQ ID NO: 2 respectively.

Table 1

LicB Polynucleotide and Polypeptide Sequences

(A) Sequences from Streptococcus pneumoniae licB polynucleotide sequence[SEQ ID NO:1].

TABLE 1 LicB Polynucleotide and Polypeptide Sequences (A) Sequences fromStreptococcus pneumoniae licB po1ynucleotide sequence [SEQ ID NO:1].5′-ATGAAAAGTAAAAACGGAGTTCCTTTTGGCCTTCTCTCAGGTATTTTCTGGGGCTTGGGTCTAACGGTTAGTGCTTATATCTTTTCGATTTTTACAGATTTGTCACCCTTTGTGGTGGCTGCAACTCATGATTTTTTGAGCATCTTTATCTTACTAGCTTTTCTCTTGGTAAAAGAAAGGAAAGTTCGCCTCTCAATTTTCTTAAATATTCGCAATGTCAGTGTTATCATAGGAGCCTTGCTAGCAGGCCCTATCGGTATGCAGGCCAATCTTTATGCAGTTAAGTATATTGGAAGTTCTTTAGCTTCATCTGTATCGGCTATTTACCCTGCGATTTCAGTTCTATTGGCTTTCTTCTTTTTGAAGCACAAGATTTCGAAAAATACTGTATTTGGGATTGTCTTGATTATTGGAGGGATTATTGCCCAGACCTATAAGGTTGAACAGGTTAATTCTTTCTACATTGGGATTCTTTGTGCTTTGGTTTGTGCTATTGCATGGGGAAGTGAGAGTGTTCTTAGCTCTTTTGCCATGGAAAGTGAATTGAGTGAAATCGAAGCCCTCTTAATCCGTCAAGTAACTTCGTTCTTGTCCTATCTTGTGATTGTGCTCTTCTCTCATCAGTCATTTGTTGCAGTAGCCAATGGACAATTGCTAGGTCTCATGATTGTCTTTGTAGCCTTTGATATGATTTCCTATTTGGCTTATTATATCGCTATCAATCGCTTGCAACCAGCCAAGGCTACAGGCTTGAACGTGAGCTATGTAGTATGGACTGTCTTGTTCGCAGCTCTTTTCTTGGGAACATCATTAGATATGCTGACCATTATGACGTCACTTGTCGTCATTGCTGGAGTTTATATTATTATTAAAGAATAA -3′ (B) Streptococcus pneumoniaelicB polypeptide sequence deduced from the polynucleotide sequence inthis table [SEQ ID NO:2].NH₂-MKSKNGVPFGLLSGIFWGLGLTVSAYIFSIFTDLSPFVVAATHDFLSIFILLAFLLVKERKVRLSIFLNIRNVSVITGALLAGPIGMQANLYAVKYIGSSLASSVSAIYPAISVLLAFFFLKHKISKNTVFGIVLIIGGIIAQTYKVEQVNSFYIGILCALVCAIAWGSESVLSSFAMESELSEIEALLIRQVTSFLSYLVIVLFSHQSFVAVANGQLLGLMIVFVAFDMISYLAYYIAINRLQPAKATGLNVSYVVWTVLFAALFLGTSLDMLTJMTSLVVIAGVYIIIKE —COOH (C)Polynucleotide sequences comprising Streptococcus pneumoniae licB ORFsequence [SEQ ID NO:3].5′-GGGAAAGTTCGCCTCTCAATTTTCTTAAATATTCGCAATGTCAGTGTTATCATAGGAGCCTTGCTAGCAGGCCCTATCGGTATGCAGGCCAATCTTTATGCAGTTAAGTATATTGGAAGTTATTTAGCTTCATCTGTATCGGCTATTTACCCTGCGATTTCAGTTCTATTGGCTTTCTTCTTTTTGAAGCACAAGATTTCGAAAAATACTGTATTTGGGATTGTCTTGATTATTGGAGGGATTATTGCCCAGACCTATAAGGTTGAACAGGTTAATTCTTTCTACATTGGGATTCTTTGTGCTTTGGTTTGTGCTATTGCATGGGGAAGTGAGAGTGTTCTTAGCTCTTTTGCCATGGAAAGTGAATTGAGTGAAATCGAAGCCCTCTTAATCCGTCAAGTAACTTCGTTCTTGTCCTATCTTGTGATTGTGCTCTTCTCTCATCAGTCATTTGTTGCAGTAGCCAATGGACAATTGCTAGGTCTCATGATTGTCTTTGTAGCCTTTGATATGATTTCATATTTGGCTTATTATATCGCTATCAATCGCTTGCAACCAGCCAAGGCTACAGGCTTGAACGTGAGCTATGTAGTATGGACTGTCTTGTTCGCAGCTCTTTTCTTGGGAACATCATTAGATATGCTGACCATTATGACGTCACTTGTCGTCATTGCTGGAGTTTATATTATTATTAAAGAATAA (D) Streptococcus pneumoniaelicB polypeptide sequence deduced from the polynucleotide ORF sequencein this table [SEQ ID NO:4].NH₂-GKVRLSIFLNIRNvSvIIGALLAGPIGMQANLYAVKYIGSYLASSvSAIYPAISVLLAFFFLKHKISKNTVFGIVLITGGIIAQTYKVEQVNSFYIGILCALVCAIAWGSESVLSSFAMESELSEIEALLIRQVTSFLSYLVIVLFSHQSFVAVANGQLLGLMIVFVAFDMISYLAYYIAINRLQPAKATGLNVSYVVWTVLFAALFLGTSLDMLTIMTSLVVIAGVYIIIKE—COOH

Deposited materials

A deposit containing a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Apr. 11, 1996 and assigned deposit number 40794. The depositwas described as Streptococcus pneumoniae 0100993 on deposit. On Apr.17, 1996, a Streptococcus pneumoniae 0100993 DNA library in E. coli wassimilarly deposited with the NCIMB and assigned deposit number 40800.The Streptococcus pneumoniae strain deposit is referred to herein as“the deposited strain” or as “the DNA of the deposited strain.”

The deposited strain contains the full length licB gene. The sequence ofthe polynucleotides contained in the deposited strain, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

One aspect of the invention there is provided an isolated nucleic acidmolecule encoding a mature polypeptide expressible by the Streptococcuspneumoniae 0100993 strain contained in the deposited strain. Furtherprovided by the invention are licB nucleotide sequences of the DNA inthe deposited strain and amino acid sequences encoded thereby. Alsoprovided by the invention are licB polypeptide sequences isolated fromthe deposited strain and amino acid sequences derived therefrom.

Polypeptides

The polypeptides of the invention include a polypeptide of Table 1 [SEQID NO:2 or 4] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of licB, and also those which have at least 70% identity to apolypeptide of Table 1 [SEQ ID NO:1 or 3] or the relevant portion,preferably at least 80% identity to a polypeptide of Table 1 [SEQ IDNO:2 or 4 and more preferably at least 90% identity to a polypeptide ofTable 1 [SEQ ID NO:2 or 4] and still more preferably at least 95%identity to a polypeptide of Table 1 [SEQ ID NO:2 or 4] and also includeportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

The invention also includes polypeptides of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the amino terminus, X is hydrogen or a metal, and at thecarboxyl terminus, Y is hydrogen or a metal, R₁ and R₃ are any aminoacid residue, m is an integer between 1 and 1000 or zero, n is aninteger between 1 and 1000 or zero, and R₂ is an amino acid sequence ofthe invention, particularly an amino acid sequence selected fromTable 1. In the formula above R₂ is oriented so that its amino terminalresidue is at the left, bound to R₁, and its carboxy terminal residue isat the right, bound to R₃. Any stretch of amino acid residues denoted byeither R group, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with licB polypeptides fragments maybe “free-standing,” or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2 or 4], or ofvariants thereof, such as a continuous series of residues that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus. Degradation forms of the polypeptides of theinvention in a host cell, particularly a Streptococcus pneumoniae, arealso preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of licB, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Streptococcuspneumoniae or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

In addition to the standard single and triple letter representations foramino acids, the term “X” or “Xaa” may also be used in describingcertain polypeptides of the invention. “X” and “Xaa” mean that any ofthe twenty naturally occurring amino acids may appear at such adesignated position in the polypeptide sequence.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the licB polypeptide havinga deduced amino acid sequence of Table 1 [SEQ ID NO:2 or 4] andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO:1 or 3], a polynucleotide of the inventionencoding licB polypeptide may be obtained using standard cloning andscreening methods, such as those for cloning and sequencing chromosomalDNA fragments from bacteria using Streptococcus pneumoniae 0100993 cellsas starting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide sequence of the invention, such as asequence given in Table 1 [SEQ ID NO:1 or 3], typically a library ofclones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E.colior some other suitable host is probed with a radiolabeledoligonucleotide, preferably a 17-mer or longer, derived from a partialsequence. Clones carrying DNA identical to that of the probe can then bedistinguished using stringent conditions. By sequencing the individualclones thus identified with sequencing primers designed from theoriginal sequence it is then possible to extend the sequence in bothdirections to determine the full gene sequence. Conveniently, suchsequencing is performed using denatured double stranded DNA preparedfrom a plasmid clone. Suitable techniques are described by Maniatis, T.,Fritsch, E. F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989). (see in particular Screening By Hybridization 1.90and Sequencing Denatured Double-Stranded DNA Templates 13.70).Illustrative of the invention, the polynucleotide set out in Table 1[SEQ ID NO:1 or 3] was discovered in a DNA library derived fromStreptococcus pneumoniae 0100993.

The DNA sequence set out in Table 1 [SEQ ID NO:1 or 3] contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 [SEQ ID NO:2 or 4] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known in the art. The polynucleotide of SEQID NO: 1, between nucleotide number 1 and the stop codon which begins atnucleotide number 877 of SEQ ID NO:1, encodes the polypeptide of SEQ IDNO:2.

LicB of the invention is structurally related to other proteins of thelicB family. See NCBI non-redundant protein database atgi|1074967|pir∥C64128 and gi|1574380 (U32829); also see TIGRStreptococcus pneumonia sequences available from The Institute forGenome Research via the internet at http://www.tigr.org/, Entry 4179;and Weiser et al., Infect. Immun., 65(3), 943-50 (1997).

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence in Table 1 [SEQ ID NO:1 or 3]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86. 821-824 (1989), or an HA tag (Wilson et al,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofcomprising nucleotide 1 to the nucleotide immediately upstream of orincluding nucleotide 877 set forth in SEQ ID NO:1 of Table 1, both ofwhich encode the licB polypeptide.

The invention also includes polynucleotides of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the 5′ end of the molecule, X is hydrogen or a metal ortogether with Y defines a covalent bond, and at the 3′ end of themolecule, Y is hydrogen or a metal or together with X defines thecovalent bond, each occurrence of R₁ and R₃ is independently any nucleicacid residue, m is an integer between 1 and 3000 or zero , n is aninteger between 1 and 3000 or zero, and R₂ is a nucleic acid sequence ofthe invention, particularly a nucleic acid sequence selected fromTable 1. In the polynucleotide formula above R₂ is oriented so that its5′ end residue is at the left, bound to R₁, and its 3′ end residue is atthe right, bound to R₃. Any stretch of nucleic acid residues denoted byeither R group, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer. Where, in apreferred embodiment, X and Y together define a covalent bond, thepolynucleotide of the above formula is a closed, circularpolynucleotide, which can be a double-stranded polynucleotide whereinthe formula shows a first strand to which the second strand iscomplementary. In another preferred embodiment m and/or n is an integerbetween 1 and 1000.

It is most preferred that the polynucleotides of the inventions arederived from Streptococcus pneumoniae, however, they may preferably beobtained from organisms of the same taxonomic genus. They may also beobtained, for example, from organisms of the same taxonomic family ororder.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae licBhaving an amino acid sequence set out in Table 1 [SEQ ID NO:2 or 4]. Theterm also encompasses polynucleotides that include a single continuousregion or discontinuous regions encoding the polypeptide (for example,interrupted by integrated phage or an insertion sequence or editing)together with additional regions, that also may contain coding and/ornon-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having adeduced amino acid sequence of Table 1 [SEQ ID NO:2 or 4]. Variants thatare fragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodinglicB variants, that have the amino acid sequence of licB polypeptide ofTable 1 [SEQ ID NO:2 or 4] in which several, a few, 5 to 10, 1 to 5, 1to 3, 2, 1 or no amino acid residues are substituted, deleted or added,in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, that do not alter the propertiesand activities of licB.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding licB polypeptide having an amino acid sequence set out in Table1 [SEQ ID NO:2 or 4], and polynucleotides that are complementary to suchpolynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding licB polypeptide andpolynucleotides complementary thereto. In this regard, polynucleotidesat least 90% identical over their entire length to the same areparticularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:1 or 3].

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in0.1×SSC at about 65° C. Hybridization and wash conditions are well knownand exemplified in Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularlyChapter 11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof; and isolating said DNA sequence. Fragments usefulfor obtaining such a polynucleotide include, for example, probes andprimers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding licB and toisolate cDNA and genomic clones of other genes that have a high sequenceidentity to the licB gene. Such probes generally will comprise at least15 bases. Preferably, such probes will have at least 30 bases and mayhave at least 50 bases. Particularly preferred probes will have at least30 bases and will have 50 bases or less.

For example, the coding region of the licB gene may be isolated byscreening using a DNA sequence provided in Table 1 [SEQ ID NO: 1 or 3]to synthesize an oligonucleotide probe. A labeled oligonucleotide havinga sequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of Table 1 [SEQ ID NOS:1 or 2 or 3 or 4] may be used inthe processes herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In addition to the standard A, G, C, T/U representations for nucleicacid bases, the term “N” may also be used in describing certainpolynucleotides of the invention. “N” means that any of the four DNA orRNA bases may appear at such a designated position in the DNA or RNAsequence, except it is preferred that N is not a base that when taken incombination with adjacent nucleotide positions, when read in the correctreading frame, would have the effect of generating a prematuretermination codon in such reading frame.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, host cells, expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic and Prognostic Assays

This invention is also related to the use of the licB polynucleotides ofthe invention for use as diagnostic reagents. Detection of licB in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also“individual(s)”), particularly mammals, and especially humans,particularly those infected or suspected to be infected with an organismcomprising the licB gene may be detected at the nucleic acid level by avariety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA, cDNA and genomic DNA may also be used in thesame ways. Using amplification, characterization of the species andstrain of prokaryote present in an individual, may be made by ananalysis of the genotype of the prokaryote gene. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the genotype of a reference sequence. Point mutationscan be identified by hybridizing amplified DNA to labeled licBpolynucleotide sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase digestion or bydifferences in melting temperatures. DNA sequence differences may alsobe detected by alterations in the electrophoretic mobility of the DNAfragments in gels, with or without denaturing agents, or by direct DNAsequencing. See, e.g., Myers et al., Science, 230: 1242 (1985). Sequencechanges at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or a chemicalcleavage method. See, e.g., Cotton et al., Proc. Nat. Acad. Sci., USA,85. 4397-4401 (1985).

Cells carrying mutations or polymorphisms (allelic variations) in thegene of the invention may also be detected at the DNA or RNA level by avariety of techniques, to allow for serotyping, for example. Forexample, RT-PCR can be used to detect mutations in the RNA. It isparticularly preferred to used RT-PCR in conjunction with automateddetection systems, such as, for example, GeneScan. RNA, cDNA or genomicDNA may also be used for the same purpose, PCR or RT-PCR. As an example,PCR primers complementary to a nucleic acid encoding licB can be used toidentify and analyze mutations.

These primers may be used for, among other things, amplifying licB DNAisolated from a sample derived from an individual. The primers may beused to amplify the gene isolated from an infected individual such thatthe gene may then be subject to various techniques for elucidation ofthe DNA sequence. In this way, mutations in the DNA sequence may bedetected and used to diagnose infection and to serotype and/or classifythe infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStreptococcus pneumoniae, comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving a sequence of Table 1 [SEQ ID NO: 1 or 3]. Increased or decreasedexpression of licB polynucleotide can be measured using any on of themethods well known in the art for the quantitation of polynucleotides,such as, for example, amplification, PCR, RT-PCR, RNase protection,Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of licB protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a licBprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

The polynucleotide sequences of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted to,and can hybridize with, a particular location on an individual microbialchromosome, particularly a Streptococcus pneumoniae chromosome. Themapping of relevant sequences to a chromosome according to the presentinvention is an important first step in correlating those sequences withgene associated with microbial pathogenicity and disease, or tochromosomal regions critical to the growth, survival and/or ecologicalniche. Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found in, for example,microbial genomic sequences available on the World Wide Web. Therelationship between genes and microbial pathogenicity, disease, or togenome regions critical to the growth, survival and/or ecological nichethat have been mapped to the same chromosomal region are then identifiedusing methods to define a genetic relationship between the gene andanother gene or phenotype, such as by linkage analysis (coinheritance ofphysically adjacent genes).

The differences in the RNA or genomic sequence between microbes ofdiffering phenotypes can also be determined. If a mutation or sequenceis observed in some or all of the microbes of a certain phenotype, butnot in any microbes lacking that phenotype, then the mutation orsequence is likely to be the causative agent of the phenotype. In thisway, chromosomal regions may be identified that confer microbialpathogenicity, growth characteristics, survival characteristics and/orecological niche characteristics.

Differential Expression

The polynucleotides and polynucleotides of the invention may be used asreagents for differential screening methods. There are many differentialscreening and differential display methods known in the art in which thepolynucleotides and polypeptides of the invention may be used. Forexample, the differential display technique is described by Chuang etal., J. Bacteriol. 175:2026-2036 (1993). This method identifies thosegenes which are expressed in an organism by identifying mRNA presentusing randomly-primed RT-PCR. By comparing pre-infection and postinfection profiles, genes up and down regulated during infection can beidentified and the RT-PCR product sequenced and matched to ORF‘unknowns’.

In Vivo Expression Technology (IVET) is described by Camilli et al,Proc. Nat'l. Acad. Sci. USA. 91:2634-2638 (1994). IVET identifies genesup-regulated during infection when compared to laboratory cultivation,implying an important role in infection. ORFs identified by thistechnique are implied to have a significant role in infectionestablishment and/or maintenance. In this technique random chromosomalfragments of target organism are cloned upstream of a promoter-lessrecombinase gene in a plasmid vector. This construct is introduced intothe target organism which carries an antibiotic resistance gene flankedby resolvase sites. Growth in the presence of the antibiotic removesfrom the population those fragments cloned into the plasmid vectorcapable of supporting transcription of the recombinase gene andtherefore have caused loss of antibiotic resistance. The resistant poolis introduced into a host and at various times after infection bacteriamay be recovered and assessed for the presence of antibiotic resistance.The chromosomal fragment carried by each antibiotic sensitive bacteriumshould carry a promoter or portion of a gene normally upregulated duringinfection. Sequencing upstream of the recombinase gene allowsidentification of the up regulated gene.

RT-PCR may also be used to analyze gene expression patterns. For RT PCRusing the polynucleotides of the invention, messenger RNA is isolatedfrom bacterial infected tissue, e.g., 48 hour murine lung infections,and the amount of each mRNA species assessed by reverse transcription ofthe RNA sample primed with random hexanucleotides followed by PCR withgene specific primer pairs. The determination of the presence and amountof a particular mRNA species by quantification of the resultant PCRproduct provides information on the bacterial genes which aretranscribed in the infected tissue. Analysis of gene transcription canbe carried out at different times of infection to gain a detailedknowledge of gene regulation in bacterial pathogenesis allowing for aclearer understanding of which gene products represent targets forscreens for novel antibacterials. Because of the gene specific nature ofthe PCR primers employed it should be understood that the bacterial mRNApreparation need not be free of mammalian RNA. This allows theinvestigator to carry out a simple and quick RNA preparation frominfected tissue to obtain bacterial mRNA species which are very shortlived in the bacterium (in the order of 2 minute halflives). Optimallythe bacterial mRNA is prepared from infected murine lung tissue bymechanical disruption in the presence of TRIzole (GIBCO-BRL) for veryshort periods of time, subsequent processing according to themanufacturers of TRIzole reagent and DNAase treatment to removecontaminating DNA. Preferably the process is optimised by finding thoseconditions which give a maximum amount of Streptococcus pneumoniae 16Sribosomal RNA as detected by probing Northerns with a suitably labelledsequence specific oligonucleotide probe. Typically a 5′ dye labelledprimer is used in each PCR primer pair in a PCR reaction which isterminated optimally between 8 and 25 cycles. The PCR products areseparated on 6% polyacrylamide gels with detection and quantificationusing GeneScanner (manufactured by ABI).

Each of these techniques may have advantages or disadvantage dependingon the particular application. The skilled artisan would choose theapproach that is the most relevant with the particular end use in mind.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunoglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-licB or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope—termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against licB-polypeptide may be employedto treat infections, particularly bacterial infections.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm “immunologically equivalent derivative” as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized”; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various formsof liposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS USA 1984:81,5849).

Antagonists and agonists—assays and molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2). Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of licBpolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bacteriocidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagoists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising licB polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a licB agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the licB polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of licB polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in licB polynucleotide or polypeptide activity,and binding assays known in the art.

Another example of an assay for licB antagonists is a competitive assaythat combines licB and a potential antagonist with licB-bindingmolecules, recombinant licB binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. LicB can be labeled, such as byradioactivity or a colorimetric compound, such that the number of licBmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing licB-induced activities, thereby preventing the action of licBby excluding licB from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of licB.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock licB protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal., Infect. Immun. 60:2211 (1992)); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial licB proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat diseases.

Helicobacter pylori (herein H. pylori) bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyfor Research on Cancer, Lyon, France;http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the international Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists oflicB) found using screens provided by the invention, particularlybroad-spectrum antibiotics, should be useful in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma. Suchtreatment should also cure gastric ulcers and gastritis.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with licB, or a fragment or variantthereof, adequate to produce antibody and/ or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Streptococcus pneumoniae infection. Also providedare methods whereby such immunological response slows bacterialreplication. Yet another aspect of the invention relates to a method ofinducing immunological response in an individual which comprisesdelivering to such individual a nucleic acid vector to direct expressionof licB, or a fragment or a variant thereof, for expressing licB, or afragment or a variant thereof in vivo in order to induce animmunological response, such as, to produce antibody and/ or T cellimmune response, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said individual from disease, whether thatdisease is already established within the individual or not. One way ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a licB or protein coded therefrom,wherein the composition comprises a recombinant licB or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid licB or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+ T cells.

A licB polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Streptococcus pneumoniae will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStreptococcus pneumoniae infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain licBprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, kits and administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Sequence Databases and Agorithms

The polynucleotide and polypeptide sequences of the invention areparticularly useful as components in databases useful for searchanalysese as well as in sequence analyisis algorithms. As used in thissection entitled Datbases and Algorithms and in claims related thereto,the terms “polynucleotide of the invention” and “polynucleotide sequenceof the invention” mean any detectable chemical or physicalcharacteristic of a polynucelotide of the invention that is or may bereduced to or stored in a computer readable form. For example,chrotatographic scan data or peak data, photographic data or scan datatherefrom, called bases, and mass spectrographic data. As used in thissection entitled Datbases and Algorithms and in claims related thereto,the terms “polypeptide of the invention” and “polypeptide sequence ofthe invention” mean any detectable chemical or physical characteristicof a polypeptide of the invention that is or may be reduced to or storedin a computer readable form. For example, chrotatographic scan data orpeak data, photographic data or scan data therefrom, and massspectrographic data.

The invention provides computer readable medium having stored thereonsequences of the invention. For example, a computer readable medium isprovided having stored thereon a member selected from the groupconsisting of: a polynucleotide comprising the sequence of apolynucleotide of the invention; a polypeptide comprising the sequenceof a polypeptide sequence of the invention; a set of polynucleotidesequences wherein at least one of said sequences comprises the sequenceof a polynucleotide sequence of the invention; a set of polypeptidesequences wherein at least one of said sequences comprises the sequenceof a polypeptide sequence of the invention; a data set representing apolynucleotide sequence comprising the sequence of ploynucleotidesequence of the invention; a data set representing a polynucleotidesequence encoding a polypeptide sequence comprising the sequence of apolypeptide sequence of the invention; a polynucleotide comprising thesequence of a polynucleotide sequence of the invention; a polypeptidecomprising the sequence of a polypeptide sequence of the invention; aset of polynucleotide sequences wherein at least one of said sequencescomprises the sequence of a polynucleotide sequence of the invention; aset of polypeptide sequences wherein at least one of said sequencescomprises the sequence of a polypeptide sequence of the invention; adata set representing a polynucleotide sequence comprising the sequenceof a polynucleotide sequence of the invention; a data set representing apolynucleotide sequence encoding a polypeptide sequence comprising thesequence of a polypeptide sequence of the invention. The computerreadable medium can be any composition of matter used to storeinformation or data, including, for example, commercially availablefloppy disks, tapes, hard drives, compact disks, and video disks.

Also provided by the inventio are methods for the analysis of charactersequences, particularly genetic sequences. Perferred methods of sequenceanalysis include, for example, methods of sequence homology analysis,such as identity and similarity analysis, RNA structure analysis,sequence assembly, cladistic analysis, sequence motif analysis, openreading frame determination, nucleic acid base calling, and sequencingchromatogram peak analysis.

A computer based method is provided for performing homologyidentification. This method comprises the steps of providing apolynucleotide sequence comprising the sequence a polynuleotide of theinvention in a computer readable medium; and comparing saidpolynucleotide sequence to at least one polynucleotide or polypeptidesequence to identify homology.

A computer based method is also provided for performing homologyidentification, said method comprising the steps of: providing apolypeptide sequence comprising the sequence of a polyptide of theinvention in a computer readable medium; and comparing said polypeptidesequence to at least one polynucleotide or polypeptide sequence toidentify homology.

A computer based method is still further provided for polynucleotideassembly, said method comprising the steps of: providing a firstpolynucleotide sequence comprising the sequence of a polynucleotide ofthe invention in a computer readable medium; and screening for at leastone overlapping region between said first polynucleotide sequence and asecond polynucleotide sequence.

A further embodiment of the invention provides a a computer based methodfor performing homology identification, said method comprising the stepsof: providing a polynucleotide sequence comprising the sequence of apolynucleotide of the invention in a computer readable medium; andcomparing said polynucleotide sequence to at least one polynucleotide orpolypeptide sequence to identify homology.

A further embodiment of the invention provides a a computer based methodfor performing homology identification, said method comprising the stepsof: providing a polypeptide sequence comprising the sequence of apolypeptide of the invention in a computer readable medium; andcomparing said polypeptide sequence to at least one polynucleotide orpolypeptide sequence to identify homology.

A further embodiment of the invention provides a computer based methodfor polynucleotide assembly, said method comprising the steps of:providing a first polynucleotide sequence comprising the sequence of apolynucleotide of the invention in a computer readable medium; andscreening for at least one overlapping region between said firstpolynucleotide sequence and a second polynucleotide sequence.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

Glossary

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Disease(s)” means and disease caused by or related to infection by abacteria, including otitis media, conjunctivitis, pneumonia, bacteremia,meningitis, sinusitis, pleural empyema and endocarditis, and mostparticularly meningitis, such as for example infection of cerebrospinalfluid.

“Host cell” is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Atschul, S. F. et al., J. Molec. Biol. 215. 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:(1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970);(2) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992); (3) Gap Penalty: 12; and(4) Gap Length Penalty: 4. A program useful with these parameters ispublicly available as the “gap” program from Genetics Computer Group,Madison Wis. The aforementioned parameters are the default parametersfor peptide comparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following: (1)Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970); (2)Comparison matrix: matches=+10, mismatch=0; (3) Gap Penalty: 50; and (4)Gap Length Penalty: 3. Available as: The “gap” program from GeneticsComputer Group, Madison Wis. These are the default parameters fornucleic acid comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO:1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO:1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 90%, 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO:2 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO:2, that is itmay be 100% identical, or it may include up to a certain integer numberof amino acid alterations as compared to the reference sequence suchthat the percent identity is less than 100% identity. Such alterationsare selected from the group consisting of at least one nucleic aciddeletion, substitution, including transition and transversion, orinsertion, and wherein said alterations may occur at the 5′ or 3′terminal positions of the reference polynucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleic acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of nucleic acidalterations for a given percent identity is determined by multiplyingthe total number of amino acids in SEQ ID NO:2 by the integer definingthe percent identity divided by 100 and then subtracting that productfrom said total number of amino acids in SEQ ID NO:2, or:

n _(n) ≦x _(n)−(x _(n) ·y),

wherein n_(n) is the number of amino acid alterations, x_(n) is thetotal number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for70%, 0.80 for 80%, 0.85 for 85% etc., · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 50, 60, 70, 80, 85, 90, 95,97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2,wherein said polypeptide sequence may be identical to the referencesequence of SEQ ID NO: 2 or may include up to a certain integer numberof amino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the integer defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of SEQ ID NO:2, that is it may be100% identical, or it may include up to a certain integer number ofamino acid alterations as compared to the reference sequence such thatthe percent identity is less than 100% identity. Such alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for70%, 0.80 for 80%, 0.85 for 85% etc., and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain Selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO:1or 3] was obtained from a library of clones of chromosomal DNA ofStreptococcus pneumoniae in E. coli. The sequencing data from two ormore clones containing overlapping Streptococcus pneumoniae DNAs wasused to construct the contiguous DNA sequence in SEQ ID NO:1. Librariesmay be prepared by routine methods, for example, using Methods 1 and 2below.

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl2351), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

4 879 base pairs nucleic acid double linear unknown 1 ATGAAAAGTAAAAACGGAGT TCCTTTTGGC CTTCTCTCAG GTATTTTCTG GGGCTTGGGT 60 CTAACGGTTAGTGCTTATAT CTTTTCGATT TTTACAGATT TGTCACCCTT TGTGGTGGCT 120 GCAACTCATGATTTTTTGAG CATCTTTATC TTACTAGCTT TTCTCTTGGT AAAAGAAAGG 180 AAAGTTCGCCTCTCAATTTT CTTAAATATT CGCAATGTCA GTGTTATCAT AGGAGCCTTG 240 CTAGCAGGCCCTATCGGTAT GCAGGCCAAT CTTTATGCAG TTAAGTATAT TGGAAGTTCT 300 TTAGCTTCATCTGTATCGGC TATTTACCCT GCGATTTCAG TTCTATTGGC TTTCTTCTTT 360 TTGAAGCACAAGATTTCGAA AAATACTGTA TTTGGGATTG TCTTGATTAT TGGAGGGATT 420 ATTGCCCAGACCTATAAGGT TGAACAGGTT AATTCTTTCT ACATTGGGAT TCTTTGTGCT 480 TTGGTTTGTGCTATTGCATG GGGAAGTGAG AGTGTTCTTA GCTCTTTTGC CATGGAAAGT 540 GAATTGAGTGAAATCGAAGC CCTCTTAATC CGTCAAGTAA CTTCGTTCTT GTCCTATCTT 600 GTGATTGTGCTCTTCTCTCA TCAGTCATTT GTTGCAGTAG CCAATGGACA ATTGCTAGGT 660 CTCATGATTGTCTTTGTAGC CTTTGATATG ATTTCCTATT TGGCTTATTA TATCGCTATC 720 AATCGCTTGCAACCAGCCAA GGCTACAGGC TTGAACGTGA GCTATGTAGT ATGGACTGTC 780 TTGTTCGCAGCTCTTTTCTT GGGAACATCA TTAGATATGC TGACCATTAT GACGTCACTT 840 GTCGTCATTGCTGGAGTTTA TATTATTATT AAAGAATAA 879 292 amino acids amino acid singlelinear unknown 2 Met Lys Ser Lys Asn Gly Val Pro Phe Gly Leu Leu Ser GlyIle Phe 1 5 10 15 Trp Gly Leu Gly Leu Thr Val Ser Ala Tyr Ile Phe SerIle Phe Thr 20 25 30 Asp Leu Ser Pro Phe Val Val Ala Ala Thr His Asp PheLeu Ser Ile 35 40 45 Phe Ile Leu Leu Ala Phe Leu Leu Val Lys Glu Arg LysVal Arg Leu 50 55 60 Ser Ile Phe Leu Asn Ile Arg Asn Val Ser Val Ile IleGly Ala Leu 65 70 75 80 Leu Ala Gly Pro Ile Gly Met Gln Ala Asn Leu TyrAla Val Lys Tyr 85 90 95 Ile Gly Ser Ser Leu Ala Ser Ser Val Ser Ala IleTyr Pro Ala Ile 100 105 110 Ser Val Leu Leu Ala Phe Phe Phe Leu Lys HisLys Ile Ser Lys Asn 115 120 125 Thr Val Phe Gly Ile Val Leu Ile Ile GlyGly Ile Ile Ala Gln Thr 130 135 140 Tyr Lys Val Glu Gln Val Asn Ser PheTyr Ile Gly Ile Leu Cys Ala 145 150 155 160 Leu Val Cys Ala Ile Ala TrpGly Ser Glu Ser Val Leu Ser Ser Phe 165 170 175 Ala Met Glu Ser Glu LeuSer Glu Ile Glu Ala Leu Leu Ile Arg Gln 180 185 190 Val Thr Ser Phe LeuSer Tyr Leu Val Ile Val Leu Phe Ser His Gln 195 200 205 Ser Phe Val AlaVal Ala Asn Gly Gln Leu Leu Gly Leu Met Ile Val 210 215 220 Phe Val AlaPhe Asp Met Ile Ser Tyr Leu Ala Tyr Tyr Ile Ala Ile 225 230 235 240 AsnArg Leu Gln Pro Ala Lys Ala Thr Gly Leu Asn Val Ser Tyr Val 245 250 255Val Trp Thr Val Leu Phe Ala Ala Leu Phe Leu Gly Thr Ser Leu Asp 260 265270 Met Leu Thr Ile Met Thr Ser Leu Val Val Ile Ala Gly Val Tyr Ile 275280 285 Ile Ile Lys Glu 290 702 base pairs nucleic acid double linearunknown 3 GGGAAAGTTC GCCTCTCAAT TTTCTTAAAT ATTCGCAATG TCAGTGTTATCATAGGAGCC 60 TTGCTAGCAG GCCCTATCGG TATGCAGGCC AATCTTTATG CAGTTAAGTATATTGGAAGT 120 TATTTAGCTT CATCTGTATC GGCTATTTAC CCTGCGATTT CAGTTCTATTGGCTTTCTTC 180 TTTTTGAAGC ACAAGATTTC GAAAAATACT GTATTTGGGA TTGTCTTGATTATTGGAGGG 240 ATTATTGCCC AGACCTATAA GGTTGAACAG GTTAATTCTT TCTACATTGGGATTCTTTGT 300 GCTTTGGTTT GTGCTATTGC ATGGGGAAGT GAGAGTGTTC TTAGCTCTTTTGCCATGGAA 360 AGTGAATTGA GTGAAATCGA AGCCCTCTTA ATCCGTCAAG TAACTTCGTTCTTGTCCTAT 420 CTTGTGATTG TGCTCTTCTC TCATCAGTCA TTTGTTGCAG TAGCCAATGGACAATTGCTA 480 GGTCTCATGA TTGTCTTTGT AGCCTTTGAT ATGATTTCAT ATTTGGCTTATTATATCGCT 540 ATCAATCGCT TGCAACCAGC CAAGGCTACA GGCTTGAACG TGAGCTATGTAGTATGGACT 600 GTCTTGTTCG CAGCTCTTTT CTTGGGAACA TCATTAGATA TGCTGACCATTATGACGTCA 660 CTTGTCGTCA TTGCTGGAGT TTATATTATT ATTAAAGAAT AA 702 233amino acids amino acid single linear unknown 4 Gly Lys Val Arg Leu SerIle Phe Leu Asn Ile Arg Asn Val Ser Val 1 5 10 15 Ile Ile Gly Ala LeuLeu Ala Gly Pro Ile Gly Met Gln Ala Asn Leu 20 25 30 Tyr Ala Val Lys TyrIle Gly Ser Tyr Leu Ala Ser Ser Val Ser Ala 35 40 45 Ile Tyr Pro Ala IleSer Val Leu Leu Ala Phe Phe Phe Leu Lys His 50 55 60 Lys Ile Ser Lys AsnThr Val Phe Gly Ile Val Leu Ile Ile Gly Gly 65 70 75 80 Ile Ile Ala GlnThr Tyr Lys Val Glu Gln Val Asn Ser Phe Tyr Ile 85 90 95 Gly Ile Leu CysAla Leu Val Cys Ala Ile Ala Trp Gly Ser Glu Ser 100 105 110 Val Leu SerSer Phe Ala Met Glu Ser Glu Leu Ser Glu Ile Glu Ala 115 120 125 Leu LeuIle Arg Gln Val Thr Ser Phe Leu Ser Tyr Leu Val Ile Val 130 135 140 LeuPhe Ser His Gln Ser Phe Val Ala Val Ala Asn Gly Gln Leu Leu 145 150 155160 Gly Leu Met Ile Val Phe Val Ala Phe Asp Met Ile Ser Tyr Leu Ala 165170 175 Tyr Tyr Ile Ala Ile Asn Arg Leu Gln Pro Ala Lys Ala Thr Gly Leu180 185 190 Asn Val Ser Tyr Val Val Trp Thr Val Leu Phe Ala Ala Leu PheLeu 195 200 205 Gly Thr Ser Leu Asp Met Leu Thr Ile Met Thr Ser Leu ValVal Ile 210 215 220 Ala Gly Val Tyr Ile Ile Ile Lys Glu 225 230

What is claimed is:
 1. An isolated polynucleotide segment comprising a nucleic acid sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the nucleic acid sequence is not genomic DNA.
 2. An isolated polynucleotide segment comprising a nucleic acid sequence of SEQ ID NO:1, wherein the nucleic acid sequence is not genomic DNA.
 3. A vector comprising the isolated polynucleotide segment of claim 1 or
 2. 4. A host cell comprising the vector of claim
 3. 5. A process for producing a polypeptide comprising the step of culturing the host cell of claim 4 under conditions sufficient for the production of the polypeptide.
 6. An isolated polynucleotide segment comprising a nucleic acid sequence that is fully complementary to a reference polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the nucleic acid sequence is not genomic DNA.
 7. An isolated polynucleotide segment comprising a nucleic acid sequence that is fully complementary to SEQ ID NO:1, wherein the nucleic acid sequence is not genomic DNA.
 8. A vector comprising the isolated polynucleotide segment of claim 6 or
 7. 9. A host cell comprising the vector of claim
 8. 